1. Field of the Invention
This invention provides for improved methods of enzymatic production of carbohydrates especially fucosylated carbohydrates. The invention provides for improved synthesis of glycosyl 1- or 2-phosphates using both chemical and enzymatic means. These phosphorylated glycosides are then used to produce sugar nucleotides which are in turn used as donor sugars for glycosylation of acceptor carbohydrates. Especially preferred herein is the use of the disclosed methods for fucosylation.
2. Summary of the Invention
This invention provides for a method of producing a fucosylated carbohydrate in a single reaction mixture comprising the steps of: using a fucosyltransferase to form an O-glycosidic bond between a nucleoside 5′-diphospho-fucose and an available hydroxyl group of a carbohydrate acceptor molecule to yield a fucosylated carbohydrate and a nucleoside 5′-diphosphate; and recycling in situ the nucleoside 5′-diphosphate with fucose to form the corresponding nucleoside 5′-diphospho-fucose. Preferred methods of this invention include the use of guanine as a base for the nucleoside, the use of catalytic amounts of nucleosides, the use of N-acetylglucosamine, galactose, N-acetylgalactosamine or N-acetyllactosamine as the carbohydrate acceptor molecule, and the use of a sialylated carbohydrate acceptor molecule.
This invention further contemplates the above method for producing fucosylated sialylated carbohydrate molecule through enzymatic formation of glycosidic linkages in a single reaction mixture comprising: forming a first glycosidic linkage between an diphosphonucleoside-activated glycosyl donor such as UDP-Gal and an available hydroxyl group of a carbohydrate acceptor molecule such as GlcNAc using a first glycosyltransferase such as β1,4-galactosyltransferase in preparing Galβ1,4GlcNAc; forming a second glycosidic linkage between a monophosphonucleoside-activated sialyl donor such as CMP-NeuAc and an available hydroxyl group of the sugar acceptor molecule such as the 3-position hydroxyl of the Gal of Galβ1,4GlcNAc using a sialyltransferase such as α2,3sialyltransferase; forming a third glycosidic linkage between a diphosphonucleoside-activated fucosyl donor such as GDP-Fuc and an available hydroxyl group of the sugar acceptor molecule such as the 3-position hydroxyl of the GlcNAc of Galβ1,4GlcNAc using a fucosyltransferase such as α1,3/4fucosyltransferase wherein at least one of steps (a) (b) or (c) further comprise the in situ formation of the phosphonucleotide-activated glycosyl donor from a catalytic amount of the corresponding monophosphate and diphosphate nucleoside. Especially preferred are methods of this invention wherein the fucosylated sialylated carbohydrate moiety product is a sialylated Lewis ligand such as sialyl Lex (SLex) or sialyl Lea (SLea) and wherein the fucose is transferred from a fucosyl donor to a hydroxyl group of a N-acetylglucosamine or galactose residue of the carbohydrate acceptor molecule.
This method embraces multiple glycosyltransferases catalyzing reactions in a single reaction mixture and preferred are those methods where one glycoslytransferase is a sialyltransferase selected from the group consisting of: α2,3 sialyltransferase, an α2,4 sialyltransferase an α2,6 sialyltransferase and α2,8 sialyltransferase. The invention contemplates the fucosylation of an oligosaccharide and preferred are those fucosyltransferases selected from the group consisting of: a α1,2 fucosyltransferase, α1,3/4 fucosyltransferase, α1,3 fucosyltransferase, α1,6 fucosyltransferase and α1,4 fucosyltransferase. Especially preferred fucosyltransferases include β-galactosidase α1,2 fucosyltransferase, N-acetylglucosamine α1,3 fucosyltransferase, N-acetylglucosamine α1,4 fucosyltransferase and N-acetyl-glucosamine α1,6 fucosyltransferase.
The carbohydrate acceptor molecules are virtually unlimited because the glycosyltransferases are not selective beyond the adjacent sugar positions. Thus they may be any carbohydrate substituted molecule wherein the carbohydrate is a Galβ1,4GlcNAc molecule or an analog thereof, or terminates in a Galβ1,4GlcNAc-X moiety and where X is an organic molecule. Additional carbohydrate acceptor molecules that are substrates for a fucoylase include analogs of Galβ1,4GlcNAc and Galβ1,4GlcNAc-X. Exemplary of such molecules as lactose, NeuAcα1,6Galβ1,4GlcNAc, Galβ1,3GlcNAc, Galβ1,4Glucal (lactal), NeuAcα2,3Galβ1,4Glucal, the 2-halo-substituted reaction products of the above glucals, Galβ1,4(5-thio)Glc, Galβ1,4GlcNAcβ-O-allyl and the like. It is to be understood that the carbohydrate acceptor molecule must contain an available hydroxyl group on the saccharide to which the donated fucosyl or other sugar group is linked, and the hydroxyl that must be present is determined by the glocsyltransferase enzyme that is utilized in the reaction.
The method contemplated herein further comprises regeneration of catalytic amounts of nucleotides used to form nucleoside sugars. A preferred bases for the nucleotides are either cytidine, guanine, or uridine. Monosaccharide donors are activated nucleotide sugars such as cytidine 5′-monophospho-N-acetylneuraminic acid, guanidine 5′-diphospho-fucose and uridine 5′-diphospho-galactose.
In addition to the above methods, this invention also contemplates in vitro reaction systems. Such systems refer to an inert or nonreactive container or compartment housing the reagents used to conduct the above described reactions. More specifically, these reaction systems have at a minimum a fucosyltransferase and a nucleoside diphosphofucose forming enzyme. These reaction systems can further comprise guanosine diphosphofucose pyrophosphorylase as the GDP-fucose-forming enzyme, a kinase such as pyruvate kinase or fructose-1,6-diphosphate kinase, acetyl kinase or fucose kinase. Other reagents can include a NADPH regeneration system or guanosine diphosphate mannose and guanosine diphospho mannose pyrophosphorylase. If a NADPH regeneration system is present it can include a catalytic amount of NADP, isopropanol in about 1 percent to about 10 percent, preferably about 2 percent to 4 percent w/v of the reaction system, and an alcohol dehydrogenase.
A number of chemical methods for synthesizing oligosaccharides are also disclosed herein. One method includes the production of a glycosyl 1- or 2-phosphate by reacting a blocked glycosyl ring having a hydroxyl at the anomeric position (1- or 2-position) with a trivalent phosphitylation reagent to yield a blocked glycosyl 1- or 2-phosphite-substituted ring. The blocked phosphite is oxidized to form a corresponding phosphate that is utilized in an enzymatic reaction. The glycosyl ring can include a galactosyl, glucosyl, fucosyl, N-acetylglucosyl and mannosyl as well as other saccharides. The preferred trivalent phosphitylating reagents are dibenzyl N,N-dialkylphosphoroamidite such as dibenzyl N,N-diethylphosphoroamidite. Such dialkyls are lower alkyls of 1-5 carbons inclusive and they can be the same or different. This method further utilizes blocking reagents such as acetyl or benzyl. The glycosyl ring is optionally from the group consisting of D- or L-aldoses having four, five or six carbons or from the group consisting of D- or L-ketoses having four, five or six carbons, as well as saccharides having up to nine carbons in the saccharide chain.
This invention further contemplates novel intermediates for the production of glycosyl 1- or 2-phosphates. A preferred intermediate is a blocked phosphityl monosaccharide of the formula I:

wherein R1 is aryl or lower alkyl;
X is independently oxygen or nitrogen;
R2 is independently an acyl, benzyl, silyl or alkyl blocking group;
R3 is independently —CH3, —OR2, —CH2OR2, —CH(OR2)—CH(OR2), or —CH(OR2)—CH(OR2)—CH(OR2);                R4 is hydrogen (H), carboxyl or C1-C5 or benzyl carboxylate ester; and        
n is 1 or 2.
In a preferred group of compounds of formula I, R4 is hydrogen so that formula I becomes formula II, below, wherein R1, R2, R3, X and n are as before defined.

One group of especially preferred compounds are those wherein the monosaccharide is a six-membered ring, R4 is H, and each X is oxygen such as mannose or fucose. Preferred are compounds wherein R1 is benzyl and R2 is benzyl or acetyl. Examples of preferred intermediates include dibenzylphosphityl 2,3,4,6-tetra-O-acetyl-D-mannoside or dibenzylphosphityl 2,3,4-tri-O-acetyl-L-fucoside.
Another group of especially preferred compounds are those wherein the monosaccharide is a six-membered ring, R1 and R2 are as above, one X is nitrogen with the others being oxygen. Exemplary compounds of that group include GlcNAc, GalNAc and NeuAc. Illustrative of these compounds are dibenzylphosphityl 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-D-glucoside and 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-D-galactoside.
A monosaccharide analog is also disclosed that is 2,3,4-tri-O-benzoyl-α-L-fucopyranosyl bromide.
Definitions
The phrase “available hydroxyl group” refers to a hydroxy-substituted carbon forming a part of the ring portion of a carbohydrate acceptor molecule that can form a glycosidic linkage through the action of a glycosyltransferase transferring a mono- or diphosphonucleoside-activated glycosyl donor to the available carbon. The “available hydroxyl group” is typically at the 3-position for fucosylation.
The phrase “blocked glycosyl ring” refers to glycosyl rings where the available amino or hydroxy substituents have been reacted with acyl, benzyl, silyl or alkyl blocking groups. Such groups have been generally described in Green, T. W., “Protective Groups in Organic Synthesis,” John Wiley and Sons, Inc., 1981.
The term “carbohydrate(s)” is meant to include any organic moiety having carbohydrates covalently linked to any monomeric saccharides. This would include disaccharides, oligosaccharides, glycolipids, glycoproteins and unnatural linkages such as saccharides bound to organic compounds not naturally bound to sugars.
The phrase “carbohydrate acceptor molecule” refers to a molecule bearing at least one monosaccharide wherein that monosaccharide has one or more available hydroxyl groups for forming glycosidic linkages with mono- or diphosphonucleoside-activated glycosyl donors.
The phrase “catalytic amount” refers to concentrations of reagents that are present in relatively minor amounts compared to reagents which are in stoichiometric amounts and are not reduced in concentration by any significant amount during the reaction process. Those reagents that are present in catalytic amounts are typically activation reagents that are then regenerated recycled into the reaction by side reactions.
The phrase “mono or diphosphonucleoside-activated glycosyl donor” or “activated donor molecule” refers to a nucleotide sugar such as uridine 5′-diphospho-galactose. These compounds contain high energy bonds that facilitate the formation of the glycosyl bond to the carbohydrate acceptor molecule. The nucleoside can be comprised any of the natural bases and sugars and can also include minor derivatives such as methyl or azo substitutions on the base, dehydroxylated or blocked hydroxy groups on the sugars, and thiophosphate analogs of the diphosphate moiety.
The phrase “glycosidic linkage” refers to a oxygen/carbon linkage typically found between sugars. It can be either α or β in its configuration and typically involves a dehydration synthesis reaction where an diphosphonucleoside-activated glycosyl donor is transferred to an available carbon of a carbohydrate acceptor molecule using a glycosyltransferase.
The phrase “glycosyl ring” refers to a sugar or amino sugar having 5 or 6 carbons in the ring. Including aldoses, deoxyaldoses and ketoses without regard for orientation or configuration of the bonds of the asymmetric carbons. This includes such sugars as ribose, arabinose, xylose, lyxose, allose, altrose, glucose, idose, galactose, talose, ribulose, xylulose, psicose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine, N-acetylneuraminic acid, fructose, sorbose, tagatose, rhamnose and fucose.
The term “glycosyltransferase” refers to a family of enzymes that join a mono- or diphosphonucleoside-activated glycosyl donor to an available carbon of a carbohydrate acceptor molecule through a glycosidic linkage. These enzymes include both enzymes purified from natural sources and sources that have been genetically modified to express such enzymes. The glycosyltransferase family includes sialyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyltransferases, fucosyltransferases, mannosyltransferases, galactogyltransferases, and KDO transferases.
The phrase “NADPH regeneration system” refers to a complement of enzymes that recycle NADP generated from an in situ enzyme reaction back to NADPH. Typically, such a system relies on an alcohol dehydrogenase converting an alcohol (isopropanol) to a ketone (acetone).
The phrase “sialylated Lewis ligand” in functional terms refers to molecule capable of binding to either the ELAM receptor or the GMP-140 receptor proteins. Chemically defined these ligands include the natural tetrasaccharide ligands SLex and SLea and derivatives thereof. Such derivatives include minor substitutions of the hydroxy groups for hydrogen, alkyl, acyloxy, alkoxy, halo, glycosyl, and the like, glycal molecules, a glycosyl ring compound in which the ring oxygen with S or NH and their alkyl, oxygenated or acyl derivatives, attachment of the anomeric carbon to carbohydrates or organic molecules, changes in the orientation and positions of glycosidic linkages or the substitution of enantiomers of the natural sugars.
The phrase “stoichiometric proportion” refers to amounts of starting product that are present in a direct proportion to the reaction products. A reagent is in stoichiometric proportion to the end products because it typically is used in the reactions producing the end product and is not regenerated during that process. Stoichiometric proportions typically approximate a 1:1 or 2:1 ratio of starting product to end product.
The phrase “trivalent phosphitylating reagent” refers to a reagent that reacts with a hydroxyl group of an organic compound to form a phosphite-containing product, which can be oxidized with an oxidizing reagent to produce a phosphate compound after deprotection.
Unless stated otherwise, all references are incorporated herein by reference.
Abbreviations
ADP, adenosine 5′-diphosphate;
ATP, adenosine 5′-triphosphate;
CMP, cytidine 5′-monophosphate;
CDP, cytidine 5′-diphosphate;
CTP, cytidine 5′-triphosphate;
CMP-NeuAc, cytidine 5′-monophospho-N-acetylneuraminic acid;
Fuc, fucose;
Fk, fucose kinase;
Fuc-1-P, fucose 1-phosphate;
Fuc-T, fucosyltransferase;
Gal, galactose;
GalNAc, N-acetylgalactosamine;
GTP, guanosine 5′-triphosphate;
GDP-Fuc, guanosine 5′-diphospho fucose;
GDP, guanosine 5′-diphosphate;
GDP-Man, guanosine 5′-diphospho-mannose;
GDP-ManPP, GDP-mannose pyrophosphorylase;
GDP-FUCPP, GDP-fucose pyrophosphorylase;
Glc-1-P, glucose-1-phosphate;
GlcNAc, N-acetylglucosamine;
ManNAc, N-acetylmannosamine;
NADP (NADPH), nicotinamide adenine dinucleotide phosphate;
NeuAc, N-acetylneuraminic acid;
NMK, nucleoside monophosphate kinase;
MK, myokinase;
PPase, inorganic pyrophosphatase;
PK, pyruvate kinase;
PEP, phospho(enol)pyruvate;
Pyr, pyruvate;
PPi, inorganic pyrophosphate;
Pi, inorganic phosphate;
Rha, rhamnose;
UDP, uridine 5′-diphosphate;
UTP, uridine 5′-triphosphate;
UDP-Glc, uridine 5′-diphospho-glucose,
UDP-Gal, uridine 5′-diphospho-galactose
Many of the structural formulas utilized herein contain only two or three groups bonded to ring carbon atoms. Following convention, the unshown groups are hydrogen atoms and are usually not depicted bonded to carbon atoms unless stereochemistry is desired to be shown. In other formulas, darkened wedge-shaped lines are used to depict bonds coming up from the plane of the page, whereas dashed wedge-shaped lines are used to depict bonds that recede from the plane of the paper. Wavy lines are used to indicate that both types of bonding (both α and β-bonds) are contemplated.